scholarly journals A High-Throughput Time-Resolved Fluorescence Energy Transfer Assay to Screen for Modulators of RGS7/Gβ5/R7BP Complex

2018 ◽  
Vol 16 (3) ◽  
pp. 150-161 ◽  
Author(s):  
Brian S. Muntean ◽  
Dipak N. Patil ◽  
Franck Madoux ◽  
James Fossetta ◽  
Louis Scampavia ◽  
...  
Keyword(s):  
Energy Transfer ◽  
High Throughput ◽  
Fluorescence Energy Transfer ◽  
Time Resolved Fluorescence ◽  
Throughput Time ◽  
Time Resolved ◽  
Fluorescence Energy

Time-Resolved Fluorescence Energy Transfer DNA Helicase Assays for High Throughput Screening

1999 ◽  
Vol 4 (5) ◽  
pp. 239-248 ◽  
Author(s):  
David L. Earnshaw ◽  
Keith J. Moore ◽  
Catherine J. Greenwood ◽  
Hakim Djaballah ◽  
Anthony J. Jurewicz ◽  
...  
Keyword(s):  
Energy Transfer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Dna Helicase ◽  
Fluorescence Energy Transfer ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Dna Helicases ◽  
Rapid Characterization ◽  
Fluorescence Energy

DNA helicases are responsible for the unwinding of double-stranded DNA, facilitated by the binding and hydrolysis of 5′-nucleoside triphosphates. These enzymes represent an important class of targets for the development of novel anti-infective agents particularly because opportunity exists for synergy with existing therapies targeted at other enzymes involved in DNA replication. Unwinding reactions are conventionally monitored by low throughput, gel-based radiochemical assays; to overcome the limitations of low throughput to achieve comprehensive characterization of adenosine triphosphate (ATP)-dependent unwinding by viral and bacterial helicases and the screening for unwinding inhibitors, we have developed and validated homogeneous time-resolved fluorescence energy transfer (TRET) assays. Rapid characterization and screening of DNA helicase has been performed in 96- and 384-well plate densities, and the ability to assay in 1536-well format also demonstrated. We have successfully validated and are running full high throughput runs using 384-well TRET helicase assays, culminating in the identification of a range of chemically diverse inhibitors of viral and bacterial helicases. For screening in mixtures, we used a combination of quench correction routines and confirmatory scintillation proximity (SP) assays to eliminate false-positives due to the relatively high levels of compound quenching (unlike other Ln3+-based assays). This strategy was successful yet emphasised the need for further improvements in helicase assays.

scholarly journals Establishment of Novel Protein Interaction Assays between Sin3 and REST Using Surface Plasmon Resonance and Time-Resolved Fluorescence Energy Transfer

2021 ◽  
Vol 22 (5) ◽  
pp. 2323
Author(s):  
Masamitsu Harada ◽  
Jun Nagai ◽  
Riho Kurata ◽  
Xiaofeng Cui ◽  
Takayuki Isagawa ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Protein Interaction ◽  
Plasmon Resonance ◽  
Fluorescence Energy Transfer ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Repressor Element ◽  
Fluorescence Energy

Repressor element-1 (RE-1) or neural restrictive silencer element (NRSE) bound with a zinc finger transcription repressor, RE-1 silencing transcription factor (REST, also known as neural restrictive silencer factor, NRSF) has been identified as a fundamental repressor element in many genes, including neuronal genes. Genes regulated by REST/NRSF regulate multifaceted neuronal phenotypes, and their defects in the machinery cause neuropathies, disorders of neuron activity), autism and so on. In REST repressions, the N-terminal repressor domain recruits Sin3B via its paired amphipathic helix 1 (PAH1) domain, which plays an important role as a scaffold for histone deacetylase 1 and 2. This machinery has a critical role in maintaining neuronal robustness. In this study, in order to establish protein–protein interaction assays mimicking a binding surface between Sin3B and REST, we selected important amino acids from structural information of the PAH1/REST complex and then tried to reconstitute it using recombinant short peptides derived from PAH1/REST. Initially, we validated whether biotinylated REST interacts with glutathione S-transferase (GST)-tagged PAH1 and whether another PAH1 peptide (PAH1-FLAG) competitively binds with biotinylated REST using surface plasmon resonance (SPR). We observed a direct interaction and competitive binding of two PAH1 peptides. Secondly, in order to establish a high-throughput and high-dynamic-range assay, we utilized an easily performed novel time-resolved fluorescence energy transfer (TR-FRET) assay, and closely monitored this interaction. Finally, we succeeded in establishing a novel high-quality TR-FRET assay and a novel interaction assay based on SPR.

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